Qubit dsdna assay kit in qubit 2.0 fluorometer

Genomic DNA was extracted from the fecal samples with the Magnetic soil and stool DNA Kit (TianGen, China) following the protocols supplied. The purity and integrity of the extracted DNA was examined using 1% agarose gel electrophoresis. The quantity of the extracted DNA was determined using Qubit^® dsDNA Assay Kit in Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA). Sterile water was used to dilute the samples to obtain an Optical Density (OD) value in the range of 1.8–2.0 for library construction. The library was prepared using the NEBNext^® Ultra DNA Library Prep Kit for Illumina (NEB, USA) following the protocol supplied with the kit. Briefly, DNA samples were randomly broken into fragments ~350 bp in length using a Covaris sonicator. The resulting fragments were subjected to end repair, A-tail and sequencing adaptor addition, purification, and PCR amplification for the library construction. Preliminary quantification was obtained using a Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA). Then the library was diluted to 2 ng/ul. The insert size of the library was detected using Agilent 2100 and Q-PCR method was used for precise quantification (the effective concentration of the library >3 nM). All libraries were then sequenced on Illumina Hiseq X10 platform with 2 × 150 bp paired reads (Novogene, Beijing, China).

Total genomic DNA was extracted from the intestinal contents (approximately 200 mg for each fish) using the QIAamp DNA extraction kit for stool (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The level of DNA degradation degree and potential contamination was monitored on 1% agarose gels. DNA concentration was measured using the Qubit® dsDNA Assay Kit in Qubit® 2.0 fluorometer (Life Technologies, CA, United States). The input material for the DNA sample preparations consisted of 1 μg DNA per sample. Sequencing libraries were generated using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, United States) following the manufacturer’s recommendations. The final 41 sample libraries of 8 groups (3 ~ 6 per group) reached grade A for metagenomic sequencing analysis. On an Illumina HiSeq platform, the library preparations were sequenced and paired-end reads were generated.

Use 1% agarose gel electrophoresis (AGE) to analyze the purity and integrity of DNA, and use Qubit^® dsDNA Assay Kit in Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA) to check DNA for quantification. Take an appropriate amount of sample into a centrifuge tube, and dilute the sample with sterile water until the OD value is between 1.8-2.0. Take 1μg genome DNA of the sample and use NEBNext^® Ultra, DNA Library Prep Kit for Illumina (NEB, USA) to construct the library. The genomic DNA was randomly sheared into fragments with a length of about 350 bp using Covaris ultrasonic crusher. The obtained fragments were end-repaired, A-tailed, and further ligated with a sequence adapter. The fragments with adapters were PCR amplified, size selected, and purified to construct the library. The constructed library was checked with Qubit2.0 for quantification, diluted to 2ng/ul, and then the insert size of the library was detected with Agilent 2100. After the insert size meets the expectation, the Q-PCR method is used to accurately quantify effective library concentration (effective library concentration is>3nM) to ensure the quality of the library. Quantified libraries will be pooled and sequenced on Illumina PE150 platforms, according to effective library concentration and data amount required.